Development and Validation of RP-HPLC Method for the Simultaneous Estimation of Paracetamol and Tramadol Hydrochloride in Tablet Dosage Form

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K.Shivaramakrishna et al / Int. J. of Pharmacy and Analytical Research Vol-3(1) 2014 [92-101]
* Corresponding author: K.Shivaramakrishna
Email:k.shivramakrishna@gmail.com
IJPAR |Volume 2 | Issue 3 | Jan - Mar -2014 ISSN: 2320-2831
Available Online at: www.ijpar.com
[Research article]
Development and Validation of RP-HPLC Method for the
Simultaneous Estimation of Paracetamol and Tramadol
Hydrochloride in Tablet Dosage Form
* 1
K.Shivaramakrishna, 2
K.Rajeswar dutt, 3
Vadthya Rajashekar
Department Of Pharmaceutical Analysis And Quality Assurance Smt. Sarojini Ramulamma
College of Pharmacy, sheshadrinagar, Mahabubnagar - 509001, Andhra pradesh, India.
.
ABSTRACT
A simple reversed-phase high-performance liquid chromatographic (RP-HPLC) method has been developed and
validated for simultaneous determination of Paracetamol and Tramadol hydrochloride in tablet dosage form.
Chromatographic analysis was performed on a Symmetry Thermo C18(150X4.6 mm,5µm) column ambient
temperature with a mixture of mixed phosphate buffer and Acetonitrile in the ratio 60:40 (mixed phosphate
buffer preparation; 0.01 M Potassium dihydrogen orthophosphate, pH 3.5 adjust with triethylamine) as mobile
phase, at a flow rate of 0.80 mL min-1
. UV detection was performed at 268 nm. The method was validated for
accuracy, precision, specificity, linearity and sensitivity. The retention times of Paracetamol and Tramadol
hydrochloride were 2.250 and 3.378 min, respectively. Calibration plots were linear over the concentration
ranges 62.500-375.000 μg mL-1
and 6.25-37.50 μg mL-1
for Paracetamol and Tramadol hydrochloride
respectively. The Limit of detection was 1.8704 and 1.254 µg mL-1
and the quantification limit was 5.6679 µg
mL-1
and 3.8008 µg mL-1
for Paracetamol and Tramadol hydrochloride respectively. The accuracy of the
proposed method was determined by recovery studies and found to be 99.56% to 100.55%.
Keywords: Paracetamol, , Tramadol hydrochloride, RP-HPLC,Validation.
INTRODUCTION
In recent times there has been an increased
tendency toward development of stabilityindicating
assays1‒ 3, using the approach to stress testing
enshrined in International conference on
harmonization (ICH) guideline Q1A (R2)4
. This
approach is being extended to drug combinations to
enable accurate and precise quantification of
several drugs in the presence of their degradation
products. Paracetamol is chemically 4-hydroxy
acetanilide (Figure 1). It is a weak inhibitor of
peripheral cyclooxygenase and its analgesic effects
may arise from inhibition of prostanoid synthesis in
the CNS. The antipyretic effects of paracetamol are
due to its action at the level of the hypothalamus to
reduce pyrogen-initiated alterations in body
temperature by inhibiting prostaglandin synthesis5 -
6.
Tramadol hydrochloric (±)-cis-2-(dimethylamino)
methyl-1- (3-methoxy-phenyl) cyclohexanol hydro
chloride (Figure 1), a synthetic analogue of
codeine, is a centrally acting analgesic agent7
. It
has been used since 1977 for the relief of severe
physical pain and has been the most widely sold
opioid analgesic drug in the world8. There are

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many reported methods for analysis of tramadol9-13
or paracetamol14-17
either alone or in combination
with other drugs18-20 in pharmaceutical dosage
forms. Very few reports are there on simultaneous
estimation of paracetamol and tramadol. They were
determined in human plasma samples using liquid
chromatography (LC-MS)21-22
. In tablets they were
estimated using spectrophotometry23-24,
HPTLC25-
26
, GC-MS27 and HPLC27-30
methods. Till date, to
the best of our knowledge, Two method has been
reported in the literature. This manuscript describes
the development and validation, in accordance with
ICH guidelines, of rapid, economical, precise and
accurate isocratic reversed-phase HPLC method for
analysis of paracetamol and tramadolHCl in table
dosage form
Figure-1 Paracetamol Figure-2 Tramadol HCl
MATERIALS AND METHODS
Chemicals
paracetamol and tramadolHCl obtained from Bio
Leo.lab.Pvt.Ltd, Hyderabad, as a gift samples.
Potassium dihydrogen orthophosphate &
Dipotassium hydrogen orthophosphate (AR Grade),
ortho-phosphoric acid (AR Grade), Acetonitrile
(HPLC Grade), were purchased from Merck (India)
Ltd., Worli, Mumbai, India. Tablet formulation
(Altracet) was purchased from local market,
containing par acetamol (325 mg), tramadol
HCl(37.5 mg). Double distilled water was used
throughout the experiment.
Instruments
Waters HPLC 2 2695 series consisting 4 pump.
Auto sampler with 5 racks, each rack has 24 vials
holding capacity with temperature control. Auto
injector has capacity to inject 5µL to 500µL. UV-
Vis Detector with PDA. Thermostat column
compartment connected it has a capacity to
maintain 5°C to 60°C column temperature.
Waters (alliance) HPLC System is equipped with
Empower-2 software.
ANALYTICAL METHOD
DEVELOPMENT
Optimization of UV conditions
Figure-3 Isobestic point of paracetamol and tramadol HCl
Chromatographic Conditions
A waters symmetry C-18 column (150 mm x 4.6
mm i.d., 5-μm) was used for chromatographic
separation. The mobile phase composed of
Acetonitrile and mixed phosphate buffer (60:40
v/v); pH adjusted to 3.5 with trietalamine at a flow
rate of 0.8 mL min-1 with run time of 8min.
Mobile phase and sample solutions were filtered
through a 0.45 μm membrane filter and degassed.
The detection of both drugs was carried out at 268
nm.
Figure-4 Optimized Chromatogram

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Fig: 4 Optimized Chromotogram
METHODOLOGY
Mobile phase preparation
Buffer preparation
0.01 M Potassium dihydrogen orthophosphate
adjust pH to 3.5 with triethylamine.
Mix buffer and Acetonitrile at 60 : 40 ratio sonicate
the resulting solution and degass it using vacuum
filtration through 0.4 micron membrane filter.
Standard stock solution preparation
Weigh and transfer 500 mg of Paracetamol
working standard and 50 mg of Tramadol
working standard into 200 mL volumetric flask,
add 50 mL of diluent and sonicate to dissolve and
dilute to volume with diluent.
Standard preparation
Transfer 10 mL of standard stock solution into
100 mL volumetric flask and dilute to volume
with diluent.
Sample Preparation
Finely grind pre weighed 20 tablets. Transfer
grinded sample quantitatively equivalent to 500
mg of Paracetamol and 50 mg of Tramadol in to
200 mL volumetric flask add 50 mL of diluent,
sonicate to dissolve for 10 minutes and dilute to
volume with diluent. Further filter the solution
through filter paper. Dilute 10 ml of filtrate to 100
ml with mobile phase.
Procedure
Inject 20 µL of blank solution, placebo solution,
Standard solution, Disregard peaks due to blank and
placebo if any.
VALIDATION OF METHOD
The HPLC method was validated in accordance
with ICH guidelines.
Precision
The system precision of the method was verified by
six replicate injections of standard solution
containing paracetamol and tramadol HCl. The
method precision ws carried out the analyte six
times using the proposed method. Repeatability
was measured by multiple injections of a
homogenous sample of paracetamol and tramadol
HCl.
Accuracy
Accuracy was carried out by % recovery studies at
three different concentration levels. To the pre-
analyzed sample solution of paracetamol and
tramadol HCl; a known amount of standard drug
powder of paracetamol and tramadol HCl were
added at 80, 100 and 120 % level.
Specificity and Selectivity
Specificity of the method was determined through
study of resolution factor of drug peak from the
nearest resolving peak. Specificity is a procedure to
detect quantitatively the analyte in presence of
component that may be expected to be present in
the sample matrix, while selectivity is the
procedure to detect qualitatively the analyte in
presence of components that may be expected to be
present in the sample matrix.
Limit of detection and Limit of quantitation
Sensitivity of the proposed method was estimated in
terms of Limit of Detection (LOD) and Limit of
Quantitation (LOQ). LOD = 3.3 x ASD/S and LOQ =
10 x ASD/S, Where, ‘ASD’ is the average standard
deviation and ‘S’ is the slope of the line.
Robustness
Robustness was evaluated by making deliberate
variations in few method parameters such as variation
of wave length; flow rate and change in mobile phase

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composition. The robustness of the method was
studied for paracetamol and tramadol HCl
RESULTS AND DISCUSSION
Selection of Chromatographic Conditions
and Optimization of Mobile Phase
Mobile phase was optimized to separate paracetamol
and tramadol HCl using Symmetry C-18 column (150
mm x 4.6 mm i.d., 5μm). Initially, ACN and phosphate
buffer in the ratio of (60:40) were tried as mobile
phase but the splitting of the peaks for both these
drugs was observed. Therefore, after adjustment of pH
of mixed phosphate buffer to 3.5 with Triethyle amine,
and mobile phase composition (ACN and phosphate
buffer in 60:40 % v/v) was tried for resolution of both
drugs. Good resolution and symmetric peaks were
obtained for both drugs when the pH of the mobile
phase (buffer) was adjusted to 3.5. The flow rate of the
mobile phase was 0.8 mL min-1. Under optimum
chromatographic conditions, the retention time for
paracetamol and tramadol HCl was found to be 2.250
and 3.378 min, respectively when the detection was
carried out at 268 nm. A typical chromatogram of two
drugs is shown in (Figure 3).
LINEARITY DATA
The Linear detector response for Paracetamol and
Tramadol hydrochloride is demonstrated by
concentration versus Area. Over the range of 25 to
150% with respect to the target concentration
(Dosage).
Table-1 For Peak Area of Paracetamol
% Conc(mcg) Area
25 62.500 456678
50 125.000 937710
75 187.500 1390513
100 250.000 1858978
125 312.500 2312992
150 375.000 2781544
Figure- 5 Calibration curve for Paracetamol
Table-2 For Peak Area of Tramadol hydrochloride
% Conc(mcg) Area
25 6.25 106154
50 12.50 217752
75 18.75 322674
100 25.00 431677
125 31.25 537365
150 37.50 647178
456678
900710
1390513
1858978
2312992
2781544
y = 18662x - 16062
R² = 0.9999
0
500000
1000000
1500000
2000000
2500000
3000000
0 50 100 150 200
Areaundercurve
Concentration(%)
LINEARITY OF PARACETMOL

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Figure-6 Calibration curve for Tramadol hydrochloride
Table-3 PRECISION
S No Name Paracetamol Tramadol HCL
RT Area RT Area
1 S-Precision-1 2.251 1851720 3.384 429022
2 S-Precision-2 2.251 1872084 3.378 433645
3 S-Precision-3 2.252 1888269 3.376 436348
4 S-Precision-4 2.251 1876113 3.372 433992
5 S-Precision-5 2.252 1898421 3.371 438659
6 S-Precision-6 2.249 1898764 3.365 438738
Average 2.251 1880895 3.374 435067
Standard Deviation 0.0011 18069.4 0.007 3680.74
RSD 0.0487 0.961 0.19 0.85
Table-4 Method Precision
S No Name Paracetamol Tramadol HC
RT Area RT Area
1 M-Precision-1 2.253 1844884 3.381 428605
2 M-Precision-2 2.252 1859494 3.379 431560
3 M-Precision-3 2.251 1880747 3.380 434968
4 M-Precision-4 2.251 1866063 3.376 433370
5 M-Precision-5 2.250 1886423 3.375 437676
6 M-Precision-6 2.252 1885501 3.372 437554
Average 2.252 1870519 3.377 433956
Standard Deviation 0.0010 16616.7 0.003 3536.26
RSD 0.0466 0.888 0.102 0.815
y = 4312x - 162.87
R² = 0.9999
0
100000
200000
300000
400000
500000
600000
700000
0 50 100 150 200
Areaundercurve
Concentration(%)
LINEARITY OF TRAMADOL

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Table-4 System Precision & Method Precision
S No Name Paracetamol Tramadol HCL
RT Area RT Area
1 S-Precision-1 2.251 1851720 3.384 429022
2 S-Precision-2 2.251 1872084 3.378 433645
3 S-Precision-3 2.252 1888269 3.376 436348
4 S-Precision-4 2.251 1876113 3.372 433992
5 S-Precision-5 2.252 1898421 3.371 438659
6 S-Precision-6 2.249 1898764 3.365 438738
7 M-Precision-1 2.253 1844884 3.381 428605
8 M-Precision-2 2.252 1859494 3.379 431560
9 M-Precision-3 2.251 1880747 3.380 434968
10 M-Precision-4 2.251 1866063 3.376 433370
11 M-Precision-5 2.250 1886423 3.375 437676
12 M-Precision-6 2.252 1885501 3.372 437554
Average 2.251 1875707 3.376 434511
Standard Deviation 0.001 17414.999 0.005 3489.901
% RSD 0.047 0.928 0.154 0.803
Result
System and Method precision
Paracetamol
% of RSD for RT = 0.047%
Area = 0.928%
Tramadol HCL
% of RSD for RT = 0.154%
Area = 0.803%
Acceptance criteria
The % of RSD for Area and RT from Repeated
injections should not be more than 2.0%.
ACCURACY
The accuracy of the test method is demonstrated by
% of recovery. The sample preparations are spiked
with known amount of standard at three
concentration levels and injected three times (Like
80% 100% and 120%).
Accuracy data.
Table-5 Standard area
S No Paracetamol Tramadol HCL
Area Area
1 1858978 431677
2 1855741 431265
Avg 1857360 431471
Table-6 Placebo
S No Paracetamol Tramadol HCL
Area Area
1 0 0
2 0 0
Avg 0 0

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Table -7 Accuracy for Paracetamol and Tramadol hydrochloride
Accuracy-- 80% Accuracy-- 100% Accuracy-- 120%
Paracetam
ol
Tramadol
HCL
Paracetam
ol
Tramadol
HCL
Paracetam
ol
Tramadol
HCL
S No Area Area Area Area Area Area
Injection-1 1492514 342654 1855965 431655 2204965 520322
Injection-2 1491210 342462 1856045 432115 2217452 520142
Injection-3 1490862 343065 1855748 430856 2234956 521345
Avg area 1491529 342727 1855919 431542 2219124 520603
amt added(mg) 400.00 40.00 500.00 50.00 600.00 60.00
amt
Recoverd(mg)
401.52 39.72 499.57 49.93 597.39 60.33
%Recovery 100.38 99.29 99.91 99.86 99.56 100.55
Results (% Of Recovery)
Paracetamol :
At 80% = 100.38 %
At 100% = 99.91 %
At 120% = 99.56 %
Tramadol HCL :
At 80% = 99.29 %
At 100% = 99.86%
At 120% = 100.55 %
Acceptance criteria
The % of recovery should be between 98 to 102%.
LIMIT OF DETECTION (LOD)
Table-8 Limit Of detection results.
S.NO Name LOD Value
(µg/ml)
1. Paracetamol 1.8704
2. Tramadol
hydrochloride
1.254
Table-9 Limit of Quantitation (LOQ)results.
S.NO Name LOQ Value(
µg/ml)
1. Paracetamol 5.6679
2. Tramadol
hydrochloride
3.8008
ROBUSTNESS
Robustness for Paracetamol and Tramadol
hydrochloride. The robustness of test method is
demonstrated by carrying out intentional method
variations like mobile phase flow changes, mobile
phase compositions and column oven temperature
variations etc...

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Table-10 Robustness for Paracetamol and Tramadol hydrochloride
S No Paracetamol Tramadol HCL
RT Area RT Area
1 Standard 2.251 1875707 3.376 434511
2 Robustness-MP-Flow Change-1 2.036 1708651 3.030 395193
3 Robustness-MP-Flow Change-2 2.517 2145545 3.756 495949
4 Robustness-Column Oven Temperature-1 2.238 1916291 3.570 443878
5 Robustness-Column Oven Temperature-2 2.323 1351173 3.146 181897
The results are mentioned below
Paracetamol
Flow1 = 2.036 min
Flow2 = 2.517 min
Temp-1 = 2.238 min
Temp-2 = 2.323 min
Tramadol HCL
Flow1 = 3.030 min
Flow2 = 3.756 min
Temp-1 = 3.570 min
Temp-2 = 3.146 min
Acceptance criteria
The result should show some variation from
standard results.
ASSAY
Assay for Paracetamol and Tramadol hydrochloride
Standard preparation
Transfer 10 ml of standard stock solution in to 100 mL
volumetric flask and make up to volume with diluent.
Sample Preparation
Transfer sample quantitatively equivalent to 500
mg of Paracetamol and 50 mg of Tramadol in to
200 mL volumetric flask add 50 mL of diluent,
sonicate to dissolve for 10 minutes and dilute to
volume with diluent. Further filter the solution
through filter paper. Dilute 10 ml of filtrate to 50
ml with mobile phase.
Procedure
Inject 20 µL of blank solution, standard solution, and
sample solution record the chromatogram. And
calculate percentage of assay.
Table-11
Paracetamol 500-mg
Tramadol HCL 50-mg
Avg wt 850-mg
Table-12
Paracetamol Tramadol HCL
S No Name RT Area RT Area
1 Standard-1 2.252 1869495 3.380 431562
2 Standard-2 2.254 1880747 3.382 434968
Avg 2.253 1875121 3.381 433265
3 Sample-1 2.253 1865425 3.378 430662
4 Sample-2 2.250 1880655 3.381 431265
Avg 2.252 1873040 3.380 430964

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Table-13 Results for Paracetamol
1873040 500 10 200 100 99.93 850 mg/Tab %Assay
1875121 200 100 850 10 100 499.45 99.89
Table- 14 Results for Tramadol hydrochloride
430964 50 10 200 100 99.84 850 mg/tab %Assay
433265 200 100 850 10 100 49.73 99.47
Assays result
Paracetamol = 99.89 %
Tramadol HCL = 99.47 %
SYSTEM SUITABILITY PARAMETERS
Table-15 System suitability parameters results for
Paracetamol and Tramadol hydrochloride
Parameters
Results
Paracetamol
Tramadol
hydrochloride
Tailing factor 1.14 1.10
Theoretical plates per
column
3621 5322
Resolution 6.24
CONCLUSION
The developed RP-HPLC method is simple,
precise, accurate, selective and reproducible. The
method has been found to be adequately rugged
and robust and can be used for simultaneous
determination of Paracetamol and Tramadol
hydrochloride in tablet formulation. The method
was validated as per ICH guidelines.
ACKNOWLEDGEMENT
The authors are thankful to Bio Leo lab.Pvt.Ltd,
Hyderabad for providing a gift samples, the authors
are also thankful to Department of pharmaceutical
analysis, Smt. Sarojini Ramulamma college of
pharmacy, Palamuru University, Mahaboobnagar,
Andhra Pradesh for encouragement
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